We have three related objectives in this proposal: 1) to characterize and elucidate the replication mechanisms of the linear genome of mitochondrial DNA from Paramecium aurelia; 2) to study the physical structure, including the location of known genes such as rRNA genes, and the genetic capacity of this mitochondrial DNA; and 3) to examine the factors involved in the transfer of mitochondrial function between closely related species. Mitochondrial DNA from paramecia replicates via lariat and dimer length molecule intermediates. Dimer length molecules are processed to two monomer length linear molecules and replication enzymes required in cleaving dimers and ligating monomers. In addition, we will determine the DNA initiation sequence as a probe for understanding mitochondrial function and evolution. The genetic capacity of the intact DNA and restriction endonuclease fragments will be examined in coupled transcription-translation in vitro systems and by analyzing the proteins made in mini cells by plasmid-cloned mitochondrial DNA. One of the most exciting aspects of Paramecium is the ability to transfer antibiotic resistance between some species (but not others) by direct microinjection of purified mitochondria. We will investigate DNA-mediated transformation, homology between species, and endonuclease and ligase activities in order to understand the successful transfers as well as the causes for "restriction" between species. It is hoped that these studies will provide fundamental information on the mechanism of replication of linear genomes a well as offer insight into mitochondrial function, biogenesis and evolution.